1. Technical Field
The present invention relates to methods and apparatus for analyzing whole blood samples, and to methods and apparatus for evaluating constituents within a whole blood sample such as white blood cells, platelets, etc.
2. Background Information
Recent advances in analytical hematology have increased the quantity and quality of information available from a patient's blood sample. As a result, the medical community's interest in using a patient's blood sample as a diagnostic tool has also increased. The methods for analyzing blood samples have not, however, in every case kept pace with the information available. Historically, blood samples have been evaluated by smearing a small amount of undiluted blood on a slide, drying, fixing and staining it, and examining the smear under a microscope. Reasonable results can be gained from such a smear, but the accuracy and reliability of the data depends largely on the technician's experience and technique. In addition, blood smears are labor intensive and cost prohibitive, and are therefore generally not favored for commercial applications.
Another known method for evaluating a whole blood sample involves diluting a volume of whole blood, placing it within a chamber, and manually evaluating the constituent cells within the diluted sample. Dilution is necessary because the number and concentration of the red blood cells (RBC's) in whole blood vastly outnumber other constituent cells. In a sample of whole blood from a typical individual, for example, there are about 4.5.times.10.sup.6 RBC's/microliter (.mu.l) of blood sample, but only about 0.25.times.10.sup.6 of platelets and 0.007.times.10.sup.6 white blood cells (WBC's) per .mu.l of blood sample. To determine a WBC count, the whole blood sample must be diluted within a range of about one part blood to twenty parts diluent (1:20) up to a dilution of approximately 1:256, depending upon the exact technique used, and it is also generally necessary to selectively lyse the RBC's with one or more reagents. Lysing the RBC's effectively removes them from view so that the WBC's can be seen. To determine a platelet count, the blood sample must be diluted within a range of about 1:100 to approximately 1:50,000. Platelet counts do not, however, require a lysis of the RBC's in the sample. A disadvantage of this method of evaluating a whole blood sample is that the dilution process is time consuming and expensive. In addition, adding diluents to the whole blood sample increases the error probability within the sample data.
A modern method for evaluating a blood sample is impedance or optical flow cytometry. Flow cytometry involves circulating a diluted blood sample through one or more small diameter orifices, each adjacent an impedance type or an optical type sensor which evaluates the constituent cells as they pass through the orifice single file. Here again, the blood sample must be diluted to mitigate the overwhelming number of the RBC's relative to the WBC's and the platelets. Although more expedient and consistent than the above described methods, flow cytometry also possesses numerous disadvantages. Some of those disadvantages stem from the plumbing required to carry the sample to, and the fluid controls necessary to control the fluid flow rate through, the sensor means. The precise control of the sample flow is essential to the operation of the flow cytometer. The plumbing within flow cytometers can and often does leak, potentially compromising the accuracy and the safety of the equipment. The fluid flow controls and dilution equipment, on the other hand, require periodic recalibration. The need for recalibration illustrates the potential for inaccurate results and the undesirable operating costs that exist with many presently available hematology analyzers which use flow cytometers. Another disadvantage is the volume of reagents required. Because of the large dilution ratios employed, correspondingly large volumes of liquid reagents are necessary. The large reagent volume increases the cost of the testing and creates a waste disposal problem.
Another approach to cellular analysis is volumetric capillary scanning as outlined in U.S. Pat. Nos. 5,547,849 and 5,585,246 for example, wherein a relatively undiluted sample of whole blood is placed into a capillary of known volume and thickness and is examined while the blood is in a quiescent state. This technique deals with the presence of the RBC's by limiting the scanning wavelengths to those with which the RBC's appear relatively transparent, and it requires that the sample be treated so that the RBC's do not aggregate during the measurement process. Thus, this technique is limited to the use of longer wavelength fluorescence, and there is no provision for the examination of RBC's and platelets or the examination of any cellular morphology.
What is needed is a method and an apparatus for evaluating a sample of substantially undiluted anti-coagulated whole blood that: 1) is capable of providing accurate results; 2) does not require removal of the RBC's prior to analysis; 3) allows the use of a wide range of light excitation sources for sample examination; 4) does not use large volumes of reagents; 5) does not require sample fluid flow during the analysis; 6) is capable of analyzing all or nearly all of the cells and particles in the sample; and 7) is cost-effective.